Amino Acid Analysis

0. Introduction

The analysis of free amino acids present in food samples, body fluids such as urine, serum and blood, and other sources, amino acid hydrolysates, from proteins, or primary and secondary amines is an important, standardized method routinely performed in biochemical, medical and biological labs. It has been, and still is used for the accurate quantification and characterization of proteins and peptides, as well as recombinant gene products. It is considered the method of choice to determine the purity and chemical composition of a protein or peptide.

1. Types of amino acid analyses

A. Hydrolysate analyses

Composition studies: Determination of amino acid content of proteins, peptides, foods, beverages, cosmetics, and others.
Quality control:             Verification of product composition

B. Physiological analyses

Nutrition studies:       Determination of free amino acid content in food supplements, and others.
Clinical assays:         Determination of amino acid content in serum, tissue, and other body fluids.

     Amino acid analysis (AAA) is a method for breaking down a protein or peptide into its components (amino acids) and determining their identities and relative quantities of the freed amino acids. Absolute quantities of amino acids released from the protein or peptide can also be determined.

     AAA can be used in combination with protein sequence analysis to verify if the total sequence of the protein in question has been determined. It also helps to identify modified amino acids like phosphor-tyrosine, the presence of amino acids modified with carbohydrates, if amino sugars are found, and others.

     Knowledge of the number of methionines or tryptophanes present allows for the design of peptide mapping strategies employing enzymatic or chemical cleavage methods to generate a limited number of longer peptides. These peptides may then be sequenced and used for the design of primers. Knowledge of the number of Lys, Arg, Asp, and Glu residues allows one to select the most appropriate protease for further experiments.

     Even today, AAA is still the most practical method for accurately quantifying amino acid, peptide and protein concentrations. Accurate measurements are essential for calculating extinction coefficients of proteins or to determine the turnover number for an enzyme. Although instrumentation performance has improved over the years, the basic concepts of amino acid analysis have not changed since Stein and Moore (1963) developed the original method based on ion exchange resins.

2. Sample preparation

     The preparation of samples to be used in AAA can be a quite difficult but very important experimental step for a successful amino acid analysis. Contamination with other proteins, amino acids, and other molecular weight solutes that interfere with the analysis chemistry are the most serious. To get accurate results the purity of the sample is much more critical than for sequencing or mass analysis.

2.1. Contamination with traces of undesired proteins:

     In the case of proteins a 10% contamination of the desired protein with an undesired byproduct such as a different protein may render the data useless.

2.2. Low molecular weight compounds

     Compounds containing primary or secondary amino groups are a very serious problem. They can be found in abundance in every laboratory and are difficult to completely eliminate. Tris and glycine are two good examples. Contamination is the major factor limiting increases in the sensitivity of analysis. The rule of thumb here is: the more steps used prior to analysis the more contamination one can pick up.

2.3. Non-amines

Non-amine compounds including many buffers, detergents, and inorganic salts, especially high salt concentrations may interfere with the analysis and need to be removed prior to analysis.

2.4. Sample treatment for physiological samples

     Physiological samples need to be treated differently than proteins prior to loading on to the derivatizer unit. To avoid losses of labile amino acids present in the sample, keep sample solutions on ice and store in freezer below -20 ºC between use or better (if enough sample is at hand), prepare aliquots and store frozen in freezer prior to analysis.

Precipitation of proteins with 5-sulfocalicylic acid (SSA):Human urine, serum and rat brain tissue extracts are treated with sulfosalicylic acid to precipitate protein: 20 µl of 35% sulfosalicylic acid is added to 200 µl of each sample. These solutions are vortexed and allowed to sit at room temperature at least 20 minutes before proceeding. The samples are then spun in a microfuge for 2 minutes and the supernatants are collected. Collagen samples are hydrolyzed in 6 N HCl, 110 oC for 24 hours. The hydrolysates are then dried down and resuspended in 250 µg/ml K4EDTA. Ant hemolymph does not need to be pretreated before analysis. Samples are loaded onto the analyzer as follows: Urine - 10 µl of a 1:2 dilution of the supernatant, Serum - 10 µl of undiluted supernatant, Rat brain extract - 10 µl of the undiluted supernatant, Collagen hydrolysate - 1.2 µg in 15 µl, Fire ant hemolymph - 4 µl of a 1:9 or 1:10 dilution. Our current knowledge that approximately 30,000 human genes appear to code for up to 1 million or more proteins has generated new interest in independent ‘de novo’ protein and peptide sequencing of gene products. Two methods are available for this task, the classical Edman chemistry based method, or the newer, more recent method which utilizes LC-MS/MS based sequencing. The second method is considered to be faster and more sensitive.

     When sufficient quantities are at hand, samples may be desalted by dialysis or size exclusion chromatography using deionized water or a volatile buffer like 1 N acetic acid. These two methods are not recommended to be used for quantities below 1 nanomole. Reversed phase-HPLC may be used for smaller sample quantities. But even this method can be tricky.

3. Hydrolysis methods

     The second part of the analysis is the hydrolysis process. Many methods have been investigated to ensure optimal recoveries for the different amino acids analyzed.

3a. Standard hydrolysis conditions are 6N HCl from 20 to 96 hours at 110 ºC in vacuo.

Limitations and recovery improving modifications of the method are listed below:

•   A single hydrolysis can not yield quantitative recoveries for all amino acids present in the sample.
•   Losses of up to 50-100% can be experienced for some amino acids.
•   Adding reductants and/or scavengers to the hydrolysis acid will improve yields of amino acids sensitive to hydrolysis conditions (ser, thr, met, tyr).
•   Shorter hydrolysis times and/or lower hydrolysis temperatures will improve yields of amino acids sensitive to hydrolysis conditions but may obscure others.
3b. Hydrolysis of sensitive amino acids

•   Serine and threonine

Side chain hydroxyl group is modified during hydrolysis (eg. esterification, dehydration). Typical losses using standard hydrolysis conditions are 15-20% for serine and 10-15% for threonine.

A typical method for quantitation is to run multiple hydrolyses at different hydrolysis times and plot the serine and threonine recovery versus length of hydrolysis (hydrolyzed for 30, 60 and 90 min). Extrapolate the recovery to time = 0 to yield an accurate quantitation.

• Tyrosine

      Typical losses are 15-20% during hydrolysis (actual losses may be higher depending on the quality of acid used and sample amount). Side chain phenol group is attacked by traces of hypochlorite/chlorine free radicals present in HCl.      Addition of scavengers is necessary to protect tyrosine. Typically phenol is added to the acid (0.1 to 1% by weight). The quality of the phenol is important. Poor quality phenol will not protect tyrosine.

• Methionine

      Losses of methionine can vary depending on sample amount, quality of HCl, amount of oxygen present in the hydrolysis vessel, length of time the hydrolyzed sample is exposed to air on the sample slide etc. Side chain thioether is oxidized forming methionine sulfone and sulfoxide.

      Addition of a reductant/scavenger improves methionine yields. Choice of reductants needs to be done carefully. Some reductants (thioglycolic acid, DTT, 2-mercapto-ethanol) react with PITC through their free sulfhydryl group and generate peaks that can interfere with the PTC-amino acid analysis. Borane-DIEA should not be used with the hydrolyzer.

• Cysteine/Cystine

      Losses can be 50% or greater. The free sulfhydryl and disulfide groups are sensitive to a variety of side reactions during hydrolysis.

      It is necessary to reduce the disulfide bonds and alkylate or oxidize the free sulfhydryl groups generated. Derivatization to form pyridylethyl or carboxy-methyl cysteine or oxidation to cysteic acid are typical techniques to quantitate this amino acid. One recommended technique is derivatization using 4-vinyl pyridine to form pyridylethyl cysteine.

• Tryptophan

      Mostly destroyed during hydrolysis by attack on the carbon double bond in the indole ring.

      It is not possible to get quantitative recovery of tryptophan from samples using acid hydrolysis. In manual hydrolysis the addition of thioglycolic acid to the acid (5-15% by volume) has given 70% recovery of trp at 500 pmol. Thioglycolic acid does generate a large interfering artifact peak in the PTC-chemistry. A method using dodecanthiol as a scavenger has proven to be useful.

• Asparagine and Glutamine

      Quantitatively recovered as aspartic and glutamic acid respectively.

Many attempts have been made over the years to improve recoveries of hydrolysis sensitive amino acids as well as to generate overall quantitative recoveries for all studied amino acids. A collection of hydrolysis conditions studied is listed in table 1.

Table 1:   Protein/Peptide Hydrolysis Methods

Method & comments	Conditions	Refs
1. Standard method	6N HCl, 110 ^oC 24-96h in vacuo
2. improved Trp, Cys, Thr, Ser, Tyr recovery	6N HCl +/- Phenol, 110^oC 20-24h in vacuo, 150^oC 1-4h in vacuo	Moore & Stein (1963)
3. improved Met, Cys, Tyr recovery	6N HCL-Na₂SO₃ , 110^oC 24h in vacuo	Swadesh et al. (1984)
4. improved Trp, Cys, Thr, Ser, Tyr recovery	HCl/Propionic acid (1:1); 150 - 160^oC 15 min, 130^oC 2h in vacuo	Westall & Hesser (1974)
5. improved Trp, Cys, Thr, Ser, Tyr recovery	HCl/TFA (2:1), 166ºC 25 min in vacuo	Tsugita & Scheffler (1982)
6. improved Trp, Cys, Thr, Ser, Tyr recovery	HCl/TFA (2:1), 5% (v/v) thioglycolic acid, 166ºC 25 min in vacuo	Yokote, et al., (1986)
7. improved Trp, recovery	6 N HCl, 0.5-6% (v/v) thioglycolic acid, 110^oC 24-64h in vacuo	Matsubara & Sasaki (1969)
8. improved Trp, recovery	3N p-Toluenesulphonic acid 110^oC 24-72 h in vacuo	Lui & Chang (1971)
9. improved Trp, recovery	3N Mercaptoethanesulfonic acid, 110^oC 24-72 h in vacuo	Penke et al., (1980)
10. Phosphoamino acids O-phospho-Ser/Thr O-phospho-Tyr O-phospho-Ser, Thr & Tyr	1-3 N NaOH, 37-50^oC 3-18 h; or 6N HCl 2 h and 4 h, respectively at 110^oC 1 h, 110^oC, 6N HCl or 5N KOH 155^oC for 30 min 6 N HCl, 110^oC 1-4 h	Kemp (1980) Martensen (1982) Capony & Demaille (1983)

4. Derivatization methods for the amino acid analysis of proteins and peptides

Many pre-column derivatization chemistries have been investigated over the years. All of them suffer from major disadvantages such as incompatibility with aqueous samples or dissolved salts, or interference from reagent peaks in the analysis chromatogram. A list of derivatization methods commonly used is shown in the next table:

Table 2: Derivatization Chemistries for Amino Acids and Amine Analysis

Method	Compound	Detection mode
1. PITC	Phenylisothiocyanate	Pre-column derivatization, UV-detection
2. OPA	Orthophthaldehyde	Pre-column derivatization, fluorescent and chemilumin-escent detection
3. FMOC-Cl	fluorenyl methyl chloroformate	Pre-column derivatization, fluorescent and chemiluminescent detection
4. OPA/FMOC-Cl combined	OPA/Fmoc-Cl	Pre-column derivatization, fluorescent and chemiluminescent detection
5. DABS	4-dimethylaminoazo-benzene-4-sulfonyl (dabsyl) chloride	Pre-column derivatization, detection: visible light
6. AQC, AccQ•Tag	6-aminoquinolyl-N-hydroxy-succinimidyl carbamate	Pre-column derivatization, fluorescent detection
7. Ninhydrin	Ruhemans purple	Post-column derivatization, detection: visible light (440 and 570 nm).

A "pre-column" derivatization method used by several companies which produce and marked amino acid analyzer typically consists of several steps as listed below:
1. Derivatization of amino acids
2. Separation of derivatized amino acids by reversed phase chromatography
3. Detection in UV or using fluorescence for increased sensitivity (some chemistries).

A "post-column" derivatization method mainly used for on-line ninhydrin derivatization in an automated amino acid analyzer consists of several steps as listed below:
1. Separation of amino acids by ion exchange chromatography
2. Derivatization of amino acids with ninhydrin at an elevated temperature
3. Detection of derivatives via absorption in the visible range (440 and 570 nm).

The ninhydrin based system has been the most widely used system.

4.1. Derivatization of free amino acids using phenylisothiocyanate (PITC)

Figure 1: Derivatization reaction of amines and free amino acids using PITC.
PITC reacts with the free amino groups in amines and amino acids to form the phenylthiourea adduct of these compounds making them suitable for UV-detection.This reaction is used in the automated hydrolyzer/derivatizer set-up with on-line HPLC separation of the resulting PTC-amino acids and UV detection. Detection is done at 268 to 270 nm.
4.2 Derivatization of free amino acids using ortho-phthalaldehyde (OPA)

Figure 2: Derivatization reaction of amines and free amino acids using ortho-phthalaldehyde (OPA).
OPA reacts with the free amino groups in aminesand amino acids in the presents of a reducing reagent like b-mercaptoethanol to form their isoindole-derivatieves making them suitable for UV-and fluorescence detection.This reaction is usually used in a pre-column derivatization step with an automated derivatizer set-up with on-line HPLC separation. UV detection is done at 338 nm. Fluorescence detection is done using excitation settings at 340 nm and emmission settings at 450 nm.
4.3. Derivatization of free amino acids using (FMOC-Cl)

Figure 3: Derivatization reaction of amines and free amino acids using 9-fluorenylmethyl-chloroformat (Fmoc).
Fmoc reacts with the free amino groups in amines and amino acids to form Fmoc-derivatieves making them suitable for UV-and fluorescence detection.This reaction is usually used in a pre-column derivatization step with an automated derivatizer set-up with on-line HPLC separation. UV detection is done at 262 nm. Fluorescence detection is done using excitation settings at 266 nm and emmission settings at 305 nm.

4.4. Derivatization using Waters AccQ•TagTM amino acid analysis system

Figure 4A: AQC (6-aminoquinolyl-N-hydroxy-succinimidyl carbamate. Chemical structure (left) and energy minimized molecular model (right). Calculations were done using the MNDO module from CACHE Scientific.

Figure 4B: Derivatization Chemistry. Both 1º and 2° amino acids and amines react rapidly with AQC to produce highly stable, fluorescent derivatives. The excess reagent reacts with water to form a free amine having significantly different fluorescence spectral properties.

References

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Amino Acid Analysis

Amino Acid Analysis

References

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